12alpha-halo-delta-pregnene-11beta, 16alpha, 21-triols and process for production thereof



Uit rates 3,660,175 IZa-IrlALG-M-REGNENE-11B,M ZI-TRIQLS AND PROCES FQR PRQJUQTEQN THEREOF Patrick A, Diassi, Westfield, Allen I. Laslrin, Somerset, and Paciiico A. Principe, South River, N.J., assignors to Olin Mathieson (Zhemical Corporation, New York,

N .Y.. a corporation of Virginia No Drawing. Filed Feb. 19, 1962, Ser. No. 174,267 18 Claims. (Cl. 260239.55)

This invention relates to the synthesis of valuable steroids and has for its objects the provision of (a) new steroids which are useful intermediates in the preparation of physiologically active steroids, and (b) an advantageous process for preparing the same.

The process of this invention comprises subjecting a steroid of the general formula:

(i1=0 O '2 Q:

wherein the 1,2-position is saturated or double-bonded to the l6a-hydroxylating action of a microorganism, such as Streptomyces roseochromogenus, thereby yielding new intermediates of this invention of the general Formula I:

CHZOH wherein the 1,2-position is saturated or double-bonded.

The l6a,2l-dihydroxy steroids are then converted to 21-halo-A -derivatives, which are also new intermediates of this invention, of the general Formula II:

CHzX

3,060,176 Patented Oct. 23, 1962 CHzOR i (rt U wherein the 1,2-position is saturated or double-bonded, and R is the acyl radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms, as exemplified by the acyl radical of an alkanoic acid of less than twelve carbon atoms (e.g., acetic, propionic, butyric, pentanoic, hexanoic and octanoic acid), a monocyclic aromatic carboxylic acid (e.g., benzoic, and p-toluic acid), a monocyclic aromatic lower alkanoic acid (e.g., phenacetic and fi-phenyl propionic acid), a monocyclic cycloalkanecarboxylic acid, a monocyclic cycloalkenecarboxylic acid, and an alkenoic acid of less than twelve carbon atoms.

These intermediates may then be hydrolyzed, as by treatment with a salt of a weak base and strong acid, such as potassium carbonate, to yield the corresponding free 21-hydroxy compounds of the general Formula IV:

(III) CHgOH g d a m CHzOY X i=0 gs 0: I I (v) wherein the 1,2-position is saturated or double-bonded, Y is hydrogen or R, wherein R is as hereinbefore defined, and X is halo.

- Alternatively, those steroids of Formula V, wherein X is fluorine can be obtained from steroids of Formula I by first esterifying the 16mand 21-hydroxy groups by reaction with the acid anhydride or acyl chloride of a hydrocarbon carboXylic acid of less than twelve carbon atoms in the presence of a base, such as pyridine, to yield the new intermediates of this invention, of the general 'Formula VI:

wherein the 1,2-position is saturated or double-bonded and R is as hereinbefore defined. The 1l1S,l2)8-oxido group is then opened with hydrogen fluoride, preferably in chloroform and ethanol or tetrahydrofuran at 30 to 10 C., to give new steroids of this invention of the general Formula VII:

CHzOR H. Wm

wherein the 1,2-position is saturated or double-bonded and R is as hereinbefore defined.

Hydrolysis of the ester groups, by the process described hereinbefore, gives the new l6u,2l-dihydroxy steroids of this invention of the general Formula VIII:

HO --OH VIII where the 1,2-position is saturated or double-bonded.

Compounds VIII are then reacted with a sulfonyl halide as described hereinbefore to yield new steroids of this invention of the general Formula IX:

4 um sulfite or hydrogen sulfide, to give the free 16u,17adihydroxy derivatives of the formula:

on on You (A 2 which are known steroids, reported to have anti-inflammatory properties.

The following examples illustrate the invention, all temperatures being in centigrade. The first three examples are directed to the preparation of starting steroids used in the process of this invention, and the remaining examples are directed to the processes of this invention.

EXAMPLE 1 115,1 219 0xido-A -PregnadieneJOFDione To a solution of 1.0 g. of 1118,12f30xidoprogesterone in 50 ml. of purified dioxane 700 mg. of 2,3-dich1oro5,6- dicyanobenzoquinone are added and the resulting solution refluxed under nitrogen for six hours. After cooling the precipitated 2,3-dichloro-5,fi-dicyanohydroquinone is filtered and washed with dioxane. The combined filtrate and washings are diluted with an equal volume of chloroform and adsorbed onto 40 g. of Woelm neutral alumina. Elution with chloroform and crystallization of the residue of the eluates from acetone-hexane gives 11/3,l2;8-0xid0 A -pregnadiene-3,20-dione.

- EXAMPLE 2 1 118,12,B-Oxidodesoxycorticosterone Surface growth from each of 2 three-week old agar slant cultures of Wojnowicia graminis NRRL 2472, the slant containing as a nutrient medium (A): glucose, 10 g.; Difco yeast extract, 2.5; K HPO 1 g.; agar, 20 g.; and distilled water to 1 'l., are suspended in 2.5 ml. of an 0.01% sodium lauryl sulfate aqueous solution. One milliliter portion of the suspension is used to inoculate four 250 ml. conical flasks, each containing 50 ml. of the following sterilized nutrient medium (B): dextrose, 10 g.; cornsteep liquor, 6 g.; NI-l I-I PO 3 g.; Difco yeast extract, 2.5 g.; CaOl 2.5; and distilled water to 1 1..

After 4 days of incubation at 25 C. with continuous rotary agitation (280 cycles per minute, 2 inch radius), 10% (vol/vol.) transfers are made to twenty 250 ml. conical flasks each containing 50 ml. of fresh sterilized medium B. After an additional incubation period of three days, 10% transfers from these flasks are made to two hundred 250 ml. conical flasks each containing 50 ml. of fresh sterilized medium B. The steroid is added by adding to each flask 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide (60 mg./ ml.) so that the medium is supplemented with 300 ug/ml. of steroid. After 48 hours of further incubation, the contents of the flasks are pooled and filtered through a Seitz clarifying pad. The flasks, mycelium and pad are washed with successive 50 ml. portions of warm water. The combined filtrate and washings have a volume of eleven liters. This is extracted with three 3.3 1. portions of chloroform which are combined, Washed twice with 5 1. portions of water and evaporated to dryness in vacuo. The residue (2.3 g.) on crystallization from acetone-hexane gives 1.0 of 11,8,l2 8-oxidodesoxycosterone having M.P. about 164-166"; [M +l79 (chlf);

is; 238 mu (e=15,200); Ag??? 2.90-3.04, 5.88, 6.02, 6.19,.

Analysis.-Calcd. for C H O (344.44): C, 73.22; H, 8.19. Found: C, 73.02; H, 8.02.

EXAMPLE 3 11B,1ZB-Oxido-A -Pregnaa'iene-Z]-Ol-3,20-Dione Following the procedure of Example 2, but substituting 11 8,128-oxido-A -pregnadiene-3,20-dione for 116,128- oxidoprogesterone in the fermentation there is obtained 11,8,12,8-oxido-A -pregnadiene-21-ol-3,20-di0ne.

EXAMPLE 4 J1,8,]ZB-Oxido-M-Pregnene-l6u,21-Di0l-3,20-Di0ne G. Soybean meal 20 Glucose 3O Soybean oil 2.2 Calcium carbonate 2.5

Distilled water to 1 liter.

The flasks are then incubated at 25 on a rotary shaker (280 cycles/minute, 2 inch radius) for 72 hours. After 72 hours, a transfer (by volume) is made to each of 47 250 ml. Erlenmeyer flasks, each containing 50 ml. of the same medium A. At the time of inoculation of these flasks, 25 mg. of steroid is added to each flask using 0.25 ml. per flask of a 100 mg./ml. solution of 1113,1Zfi-oxidodesoxycorticosterone in N,N-dimethylformamide. A total of 1.09 g. of steroid is thereby fermented. After inoculation and supplementation the flasks are then incubated under the same conditions as described above. At approximately 70 hours after steroid addition, the culture broths are harvested. The contents from each flask are pooled and the pooled broth is then adjusted to pH 4.5 using 12 N H 50 The acidified broth is then filtered through a Biichner-Seitz clarifying pad apparatus. The filtrate (3000 ml.) is collected and extracted three times with one liter portions of chloroform. The combined chloroform extracts are washed twice with 1.5 l. of water and then evaporated to dryness in vacuo. Crystallization of the residue from acetone-hexane give about 300 mg. of 11,8,12/3-oxido-A -pregnene-16a,21-diol-3,20 dione having M.P. about 212-214"; [a] +158 (chlf.);

653". Analysis.Calcd. for C H O (360.44): C, 69.97; H, 7.83. Found: C, 70.14; H, 8.04.

EXAMPLE 5 1J13,12,8-Oxido-A -Pregnadiene-l60,21-Di0l-3,20-Di0ne Following the procedure of Example 4, but substituting an equivalent amount of 11fi,12/3-oxido-A -pregnadiene-21-ol-3,20-dione for the 11B,12;8-oxido-A -pregnene- 21-ol-3,20-dione in the fermentation, there is obtained 115,12}3-oxido-A -pregnadiene-16u,21-diol-3,20-di0ne.

EXAMPLE 6 116,1ZB-Oxido-M-Pregnene-Z6a,21-Di0l-3,20-Di0ne 16,217-Diacelate tion of the residue from acetone-hexane gives 160 mg. of 11B,12,8-oxido-A -pregnene-l6a,21-diol-3,20-dione 16, 21-diacetate having M.P. about 187-188" +144 (chlf.),

:13.. 238 mp (e=l7,500)

Analysis.Calcd. for C25H32O7.H2O (462.53): C, 64.92; H, 7.41. Found: C, 65.28; H, 7.30.

EXAMPLE 7 1 16,1 2,8-0xid0-A -Pregnadien e-I 60,21-Di0l-3,20-D ione 1 6,21 -Diacetate Following the procedure of Example 6 but substituting 11 ,8,1ZB-oxido-A -pregnadiene-16a,21-diol-3,2O-dione for 11B,12,8-oxido A pregnene-16ot,21-diol-3,20-dione there is obtained 11B,1Zfl-oxido-A -pregnadiene-160:,21- diol-3,20-dione 16,21-diacetate.

EXAMPLE 8 IZa-FluOro-M-Pregnene-J 1[3,]6a,21-Tri0l-3,20-Di ne 1 6,21-Diacetate To a stirred solution of 125 mg. of 11/8,12fl-oxido-A pregnene-l6a,21-diol-3,20-dione 16,21-diacetate in'5 ml. of chloroform and 1.2 ml. of tetrahydrofuran contained in a polyethylene bottle and cooled in an acetone-Dry Ice bath 1 ml. of hydrogen fluoride is added. After 30 minutes the acetone-Dry Ice bath is replaced by an ice bath and the mixture stirred at 0 C. for 5 hours. It is then poured into a mixture of ml. each of chloroform and ice water contained in a polyethylene beaker and neutralized with NaHCO with stirring. The chloroform is separated Washed with water and evaporated to dryness, in vacuo. The residue is chromatographed on 3 g. of Woelm neutral alumina and elution With chloroformbenzene (12B) followed by crystallization of the residue from acetone-hexane gives 41.2 mg. of 12oc-flll0IO-A pregnene-1lfl,16a,21-triol-3,20-dione 16,21-diacetate having M.P. about 185187, [M +153 (chlf.);

ii 238 mu (216,900)

Analysis.Calcd. for C H O F (464.51): C, 64.64; H, 7.20; F, 4.09. Found: C, 65.11; H, 6.99; F, 4.54.

EXAMPLE 9 1 2 a-F lu0r0-A -Pregnadiene-1 I ,B,] 6 (1,21 -T rial-3 ,20- Diane 16,21-Dz'acetate Following the procedure of Example 8 but substituting 115,1ZB-oxido-A -pregnadiene-16a,21-diol-3,20-dione 16,21-diacetate for 1lfl,IZB-oxido-M-pregnene-l6a,21-diol-3,20-dione 16,21-diacetate there is obtained 12a-fluoro- A -pregnadiene-1 1B, 160:,21-t1i01-3 ,20-dione 16,21-diacetate.

EXAMPLE 10 JZa-FluorO-N-Pregnene-J]/3,16u,21-Tri0l-3,20-Di0ne EXAMPLE 1 1 I2 a-Fluoro-A -Pregnadiene-1 113,1 611,2] -Tri0l-3,20-Dione Following the procedure of Example 10 but substituting 12a,fluoro-A -pregnane-1113,16a,21-triol-3,20-dione- 1 6,21diacetate for 12a-fluoro-A pregnene-11fi,16 x,21-triol-3,20-dione 16,21-diacetate there is obtained c-fil1010- A -pregnadiene-l1B,l6a,21-triol-3,20-dione.

7, EXAMPLE 12 1 1 6,1 2 {3-Oxid0-21 -Chlore-A -Pregnadiene-3J0-Dione.

To a cold solution of 240 mg. of 11B,12;8'oxido-A pregnene-16a,21-diol-3,20-dione in 15 ml. of dry pyridine, 0.6 ml. of methanesulfonyl chloride is added dropwise. The reaction mixture is stoppered and keptat 5 C. for 2 hours following which crushed ice is added'to decom pose the excess methanesulfonyl chloride. The mixture is then distributed between chloroform and Water. The chloroform is separated, washed successively with 2 N HCl, 5% raHcO and water and evaporated to dryness in vacuo. The residue is chromatographed on 6 g. of Florisil. Elution with benzene, evaporation of the solvent in vacuo and crystallization from acetone-hexane gives 100 mg. of 116,126-xido-21-chloro-A -pregna diene-3,20-dione having M.P. about l82184; [@15 +240 (chli);

Analysis.-Calcd. for C H O C1 (360.86): C, 69.89; H, 6.98; Cl, 9.83. Found: C, 70.77; H, 7.28; Cl, 9.77.

' EXAMPLE 13 1 1 6,125-0xid0-21 -ChZora-A -Pregnatriene-3,ZO-Dione Following the procedure of Example 12, but substituting 115,12fi-oxido-A -pregnadiene-16e,21-diol-3,20-dione for the 11B,12;S-oxido- -pregnene-16a,21-diol-3,20-dione there is obtained 116,12,8-oxido-21-chloro-A -pregnatriene-3,20-dione.

EXAMPLE 14 1 2 a-F [1101-0-21 -Chloro-A -Pregnadiene-1 1,8-01- 3,20-Di0ne Following the procedure of Example 12, but substituting 12oz-fll101'0 A pregnene-l16,16u,21-triol-3,ZO-dione for the 11B,l2t3-oxido-A -pregnene-l6a,21-diol-3,20-dione there'is obtained 12oc-fluoro-21-chloro-A -pregnadiene- 1 11B-ol-3,20-dione.

EXAMPLE 15 1Zm-FluorO-ZI-Chlore-A -Pregnatriened 113-0!- 7 3,20-Di0ne Following the procedure ofExample 12, butsubstituting 12u-fiuoro-A -pregnadiene-1lfl,16u,21-triol-3,20-dione for the 1113,12fi-oxido-M-pregnened6e,21-diol-3,20- dione there is obtained 12a-fluoro-2l-ch1oro-A -prcgnatriene-l 16-ol-3,20-dione.

EXAMPLE 16 1 1[3,1213-Oxia'0-A -Pfegnadiene-21 -Ol-3,20-Di ne 21 -A cetate tration of the solution in vacuo crystals separate which are filtered, Washed with water and dried to give 638 mg. of 116,12,8-oxido-A -pregnadiene-21-ol-3,20'dione 21 acetate, M.P. about 170l71; [M +228 (chlfi);

A213... 3 my ;l:;? 5.72, 5.98, 6.01, 6.19,- 6.31..

Analysis.-Calcd. for (3 1-1 0 (384.45): C, 71.85; H, 7.34. Found: C, 71.94; H, 7.27.

EXAMPLE 17 1 16,1ZB-Oxido-A -Pregnatrien-Z1-0l-3,20-Di0ne 21 Acetate Following theprocedure of Example 16 but substituting 11/3,126-oxido-21-chloro-A -pregnatriene,20-dione for the l1p,12,8-oxido-21-chloro-A -pregnadieue-3,

8?- 20-dione, there is obtained 11B,12;3-oxido-A -pregnatriene-21-o1-3,20-dione ZI-acetate.

EXAMPLE 18 I Z a,F l Hora-A -Pregnadiened 1;8,21-Di0l-3,20-Dione 21 -Acetate Following the procedure of Example 16'but substituting 12oc-fluo1'o-21-chloro-A -pregnadiene-1lfi-ol-3,20-dione for the 1113,125-oxido-21-chloro-A -pregnadiene-3, 20-dione, there is obtained 12a-fluoro-A -pregnadiene- 1lfi,21-diol-3,20-dione 21-acetate.

EXAMPLE 19 1 2 u-F Imam-A -Pregnatriene-1 1 B,21-Di0l-3,20-Di0ne 21 Acetate Following the procedure of Example 16, but substitut ing 12ix-fiuoro-2 l-chloro-A -pregnatriene-1 1,8-ol-3;20- dione for the 115,125-oxido-21-chloro-A*' -pregnadiene- 3,20-d1'one, there is obtained 12a-fluoro-A -pregnatriene-l1B,21-diol-3,20-dione 21-acetate.

EXAMPLE 20 1 113,12fi-Oxido-M -Pregnadiene-21 -0l-3,20-Di0ne Following the procedure of Example 10, but substituting 11,3,l26-oxido-A regnadien-Z1-ol-3,ZO-dione 21- acetate for the 12oc-fiuoro-A -pregnene-11fl,l6a,21-triol- 3,20-dione 16,21-diacetate, there is obtained 116,126- oxido-A -pregnadiene-2l-ol-3,20-dione.

Similarly, 11 8,1219 oxido-A -pregnatriene-21-ol-3, 20-dione ZI-acetate, 1Zea-duoro-A -pregnadiene-116,21- diol-3,20-dione 21-acetate and 1Za-fiuOrQ-A regnatriene-l1B,21-diol-3,20-dione 21-acetate are hydrolyzed to their respective 'll-hydroxy derivatives.

EXAMPLE 21 12 e-F Moro-A -Pregnadiene-1 1B,21-Di0l-3,20-Dione 21-Acetate To a stirred solution of 64.4 mg. of 11B,12,8-oxido- A -pregnadiene-21-ol-3,20-dione 21-acetate in 5 ml. of chloroform and 1.2 ml. of tetrahydrofuran contained in a polyethylene bottle and cooled by means of an acetone- Dry Ice bath, 1.0 ml. of hydrogen fluoride is pipetted by means of a polyethylene pipette. The reaction vessel is stoppered and left at 5 C. for 5 hours andthen- EXAMPLE 22 12a-Flu0ro-A -Pregnatriene-1 1B,21-Di0l-3,20-Di0ne 21 -Acetate Following the procedure of Example 21, but substituting 11,6,12fl-oxido-A fi' -pregnatriene-Z1-o1-3,20-dione 21-acetate for 116,12,8-oxido-A -pregnadiene-Z1-ol-3,20- dione 21-acetate, there is obtained l2e-fluoro-A pregnatriene-l1B,21-diol-3,20-dione 21 acetate.

EXAMPLE 23 12a-Chloro-A -Pregnadiene-1 1 [3,21-Di0l-3,20-Di0ne ZI-Acetate Following the procedure of Example 21 but substituting hydrogen chloride for the hydrogen fluoride, 12achloro-A -pregnadiene-115-21-diol-3,20-dione 21-acetate is obtained.

Similarly, hydrogen bromide yields 12a-bromo-A pregnadiene-l15,2l-diol-3,20-dione 2l-acetate and hydrogen iodide yields 12a-iodo-A -pregnadiene-115,21-diol- 3,20-dione 21 acetate.

EXAMPLE 24 IZa-FZZIOIO-I 6 oc-H ydrxyhydr0c0rtis0ne-2I -A cemte To a stirred solution of 25.4 mg. (0.063 mmol) of 1204 fluoro A4116 pregnadiene 115,21 diol 3,20- dione 21-acetate in 4 m1. of benzene containing 0.2 ml. of pyridine a solution of 16.7 mg. (0.066 mmol) of osmium tetroxide in 0.82 ml. of benzene is added dropwise over a ten minute period. The solution is stirred at room temperature and after 45 minutes a precipitate separates. The stirring is continued for 2 /2 hours and then 4 ml. of an aqueous solution containing .36 g. each of sodium sulfite and potassium bicarbonate which is free of oxygen is added followed by 2 ml. of methanol. The mixture is then stirred under nitrogen for 3 hours, filtered, and washed thoroughly with chloroform and warm tetrahydrofuran. The filtrate is Washed with water and evaporated to dryness in vacuo. Crystallization of the residue from acetone-hexane gives 10 mg. of 120:- fluoro-16x-hydroxyhydrocortisone 21-acetate, M.P. about 204206; [0:] +101 (chlf.);

A533; 2939 m (e=l6,500); 2.76, 2.88, 3.00, 5.80, 6.03,

max.

Analysis.Calcd. for C H O F (438.48): C, 62.99; H, 7.13. Found: C, 62.38; H, 7.57.

EXAMPLE 25 12a-Flz10r0-16a-Hydroxyprednis0lone 21-A cetate Following the procedure of Example 24 but substituting 12u-fluoro-A -pregnatriene-115,2l-diol-3,20-di- One 21-acetate for 12a-fluQrmA -pregnadiene-115,21- diol-3,20-dione 21-acetate there is obtained 12oz-fluoro- 16a-hydroxyprednisolone 21-acetate.

This invention may be variously otherwise embodied within the scope of the appended claims.

What is claimed is:

l. A compound selected from the group consisting of steroids of the general formula OH2OY pregnene 160:,21 diol 3,20-

- pregnadiene 16a,21 diolpregnene 160:,21 diol 3,20-

- pregnadienegroup consisting of OHzOY and the 1,2-dehydro derivatives thereof, wherein Y is selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms.

8. 115,125-oXido-A -pregnadiene-Zl-ol-3,20-dione 21- acetate.

9. 115,125-0Xid0-A -pregnadiene-21-ol-3,20-di0ne.

10. 115,125 oxido A pregnatriene 21 o1 3, 20-dione 21-acetate.

11. 115,125 oxido A1416 20-dione.

12. A compound selected from the group consisting of l2ot-fluoro-2l-halo-A -pregnadiene--ol-3,20-dione and .12rx-fluoro-21halo-A -pregnatriene-115-ol-3,20- dione, wherein the 21-halo is selected from the group consisting of chlorine, bromine and iodine.

13. l2a-fluoro-21-chloro-A -pregnadiene-1 15-ol-3,20- dione.

14. A compound selected from the group consisting of steroids of the general formula pregnatriene 21 ol -3,

OH OY and the 1,2-dehydro derivatives thereof, wherein Y is selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms.

15. 12a-fluoro-A -pregnene-'115,16w,21-ttriol-3,20-dione 16,21-diacetate.

16. 12oc-fluoro-A -pregnene-115,16u,21-triol-3,20-dione.

17. A process for preparing a steroid of claim 5 which comprises interacting a corresponding dihydroxy steroid of claim 1 with a sulfonyl halide in the presence of a base and recovering the 16-dehydro-21-halo steroid formed.

18. A process for preparing a steroid of claim 7 which comprises interacting a corresponding steroid of claim 5 with a salt of a hydrocarbon carboxylic acid of less than twelve carbon atoms in the presence of an alkali metal iodide and recovering the 21-ester formed.

No references cited.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,060,176 October 23, 1962 Patrick A, Diassi et a1.

It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 9, line 27, for a] read [@13 lines 47 to 58, the formula should appear as shown below instead of as in the patent:

ea er Signed and sealed this 2nd day of April 1963.

(SEAL) Attest:

ESTON GB JOHNSON DAVID L, LADD Attesting Officer Commissioner of Patents 

14. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF STEROIDS OF THE GENERAL FORMULA 